Picornaviruses are a diverse family of viruses which cause a number of common illnesses. Of the Picornaviridae family, viruses of the genus Enterovirus, which are all very closely related, are significant for the number of diseases they cause.
Viruses of the genus Enterovirus affect millions of people worldwide each year, and are often found in the respiratory secretions (e.g., saliva, sputum, or nasal mucus) and stool of an infected person. Enterovirus infects the gut, thus the derivation of their name from the root “enteric”. Historically, poliomyelitis was the most significant disease caused by an enterovirus, that is, poliovirus. There are 62 non-polio enteroviruses that can cause disease in humans: 23 Coxsackie A viruses, 6 Coxsackie B viruses, 28 echoviruses, and 5 other enteroviruses. Polioviruses, as well as Coxsackie viruses and echoviruses, are spread through the fecal-oral route. Infection can result in a wide variety of symptoms ranging from mild respiratory illness (common cold), hand, foot and mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis.
Of the picornaviruses, Enterovirus represents a genus of a large and diverse group of small RNA viruses characterized by a single positive-strand genomic RNA. All enteroviruses contain a genome of approximately 7,500 bases and are known to have a high mutation rate due to low-fidelity replication and frequent recombination. After infection of the host cell, the genome is translated in a cap-independent manner into a single polyprotein, which is subsequently processed by virus-encoded proteases into the structural capsid proteins and the nonstructural proteins, which are mainly involved in the replication of the virus.
The enteroviruses are associated with several human and mammalian diseases. Serologic studies have distinguished 66 human Enterovirus serotypes on the basis of antibody neutralization tests. Additional antigenic variants have been defined within several of the serotypes on the basis of reduced or nonreciprocal cross-neutralization between variant strains. On the basis of their pathogenesis in humans and animals, enteroviruses were originally classified into four groups, polioviruses, Coxsackie A viruses (CA), Coxsackie B viruses (CB), and echoviruses, but it was quickly realized that there were significant overlaps in the biological properties of viruses in the different groups.
The Enterovirus genus includes the following ten species:                Bovine enterovirus         Human enterovirus A        Human enterovirus B        Human enterovirus C        Human enterovirus D        Human rhinovirus A        Human rhinovirus B        Human rhinovirus C        Porcine enterovirus B        Simian enterovirus A        
Within these ten species are there are various serotypes, for example:                Enterovirus serotypes HEV71, EV-76, EV-89, EV-90, EV-91, EV-92 and Coxsackievirus A16 are found under the species Human enterovirus A.        Serotypes Coxsackievirus B1 (CV-B1), CV-B2, CV-B3, CV-B4, CV-B5 (incl. swine vesicular disease virus [SVDV]), CV-B6, CV-A9, echovirus 1 (E-1; incl. E-8), E-2, E-3, E-4, E-5, E-6, E-7, E-9 (including CV-A23), E-11, E-12, E-13, E-14, E-15, E-16, E-17, E-18, E-19, E-20, E-21, E-24, E-25, E-26, E-27, E-29, E-30, E-31, E-32, E-33, enterovirus B69 (EV-B69), EV-B73, EV-B74, EV-B75, EV-B77, EV-B78, EV-B79, EV-B80, EV-B81, EV-B82, EV-B83, EV-B84, EV-B85, EV-B86, EV-B87, EV-B88, EV-B93, EV-B97, EV-B98, EV-B100, EV-B101, EV-B106, EV-B107, EV-B110 (from a chimpanzee) and the simian enterovirus SA5, are found under the species Human enterovirus B.        Serotypes EV-95, EV-96, EV-99, EV-102, EV-104, EV-105, and EV-109 are found under the species Human enterovirus C.        Serotypes EV-68, EV-70, & EV-94 are found under the species Human enterovirus D.        Poliovirus serotypes PV-1, PV-2, and PV-3 are found under the species Human enterovirus C.        
Diseases caused by enterovirus infection include poliomyelitis which is the most notable disease caused by an enterovirus infection. Nonspecific febrile illness is, however, the most common presentation of an enterovirus infection.
Enteroviruses are the most common causes of aseptic meningitis in children. In the United States, enteroviruses are responsible for 30,000 to 50,000 cases of meningitis. Encephalitis is a rare manifestation of an enterovirus infection; when it occurs, the most frequent Enterovirus found to be causing the encephalitis is echovirus 9.
Pleurodynia caused by enteroviruses is characterized by severe paroxysmal pain in the chest and abdomen, along with fever, and sometimes nausea, headache, and emesis.
Pericarditis and/or myocarditis are typically caused by enteroviruses. Arrythmias, heart failure, and myocardial infarction have also been reported.
Acute hemorrhagic conjunctivitis can be caused by enteroviruses.
Hand, foot and mouth disease is a childhood illness most commonly caused by infection by Coxsackie A virus or HEV71.
A 2007 study suggested that acute respiratory or gastrointestinal infections associated with enteroviruses may be a factor in chronic fatigue syndrome.
All members of the genus Enterovirus, including HEV71, polioviruses and Coxsackievirus A16 have a single stranded positive sense RNA genome which has a single open reading frame encoding a polyprotein, P1, consisting of the capsid proteins VP4, VP2, VP3 and VP1 and several non-structural proteins including the viral proteases 3C and 3CD which are responsible for cleaving the polyprotein P1 into individual capsid proteins VP1, VP3 and VP0, which VP0 is eventually cleaved into VP2 and VP4. The capsid proteins may assemble into virus like particles (VLPs).
Human enterovirus 71 (HEV71) and Coxsackievirus A16 are Enterovirus serotypes notable as the major causative agents for hand, foot and mouth disease (HFMD), and HEV71 is sometimes associated with severe central nervous system diseases. HEV71 was first isolated and characterized from cases of neurological disease in California in 1969. To date, little is known about the molecular mechanisms of host response to HEV71 infection, but increases in the level of mRNAs encoding chemokines, proteins involved in protein degradation, complement proteins, and pro-apoptotic proteins have been implicated.
Hand Foot and Mouth Disease (HFMD) is a common, self-limiting illness of children caused by a group of species A enteroviruses (Picornaviridae family) such as human Coxsackievirus A16 (CVA16), Coxsackievirus A10 (CVA10) and Human enterovirus A 71 (HEV71). The virus is excreted in feces and is also found in pharyngeal secretions. Transmission is associated with close contact among children and through environmental contamination. The disease is characterized by an acute onset of fever with a rash on the palms, soles, buttocks, and knees, and vesicles on buccal membranes that usually resolve in 7-10 days. Only a small proportion of children with HFMD develop severe disease.
Severe disease involving primarily the neurologic and cardiovascular systems manifesting as syndromes such as meningitis, encephalitis, acute flaccid paralysis, pulmonary edema and cardiac failure generally occur only with HEV71 infection. In the Asia-Pacific Region the most devastating neurological syndrome is brainstem encephalitis, which has a mortality rate of 40-80 percent. Children with severe HFMD may take months to recover, and in some cases the neurologic damage may be permanent. Currently, there is no specific antiviral treatment for HFMD and no vaccines to prevent enterovirus infection other than polio.
HEV71 was first isolated from a child who died of encephalitis in California in 1969, and first reported in 1974. Although the virus has been detected worldwide since then, the recent regional epidemics of HFMD in Asia has raised concern that more pathogenic forms of HEV71 may be emerging in the region. The first recognition of a HFMD outbreak with a high number of fatalities was in Sarawak, Malaysia in 1997. The virus associated with the outbreak then was HEV71. Taiwan reported 129,106 HFMD cases in a 1998 epidemic with 405 having severe disease with 78 deaths. Singapore reported an epidemic of 9000 cases with 7 deaths during 2000-2001, and since then has experienced recurrent epidemics every two to three years. During the first 8 months of 2008, Singapore reported 19,530 cases and one death due to HFMD. Since then HEV71 outbreaks have been reported regularly in Singapore, Thailand, Malaysia, Taiwan, Japan, Korea and Vietnam.
China reported 83,344 cases with 17 deaths in 2007, and in 2008 experienced a large outbreak in Fuyang City in Anhui Province spreading throughout many parts of China. These large outbreaks were widely covered by the press, which highlighted parental concerns about the health of their children and the social disruption from closing of schools and day care centers by public health departments in an attempt to break the chain of transmission. Since then China has reported large outbreaks annually.
With regard to disease caused by other members of the Picornaviridae family, natural infection and prevalence of polio have occurred exclusively in the human being since ancient times as an infectious disease. A large number of humans still become infected with polio every year in developing countries. Hence, the eradication of polio is an ongoing process.
Polioviruses were formerly classified as a species belonging to the genus Enterovirus in the family Picornaviridae. The Poliovirus species has been eliminated from the genus Enterovirus. The poloviruses are classified as serotypes, Human poliovirus 1 (PV-1), Human poliovirus 2 (PV-2), and Human poliovirus 3 (PV-3), and are considered to be subtypes of species Human enterovirus C, in the genus Enterovirus in the family Picornaviridae. The type species of the genus Enterovirus was changed from Poliovirus to Human enterovirus C in 2008.
The three subtypes of species Human enterovirus C, PV-1, PV-2 and PV-3, are characterized by a slightly different capsid protein. Capsid proteins define cellular receptor specificity and virus antigenicity. PV-1 is the most common form encountered in nature; however, all three forms are extremely infectious and can affect the spinal cord and cause poliomyelitis.
Infection with Human enterovirus C has been a widespread problem and inactivated whole virus vaccines have been used for mass immunization and are currently available. Good results have been obtained with inactivated poliomyelitis vaccines which may be prepared according to a method which has been developed by Salk and has been improved later in several aspects. Generally, these vaccines contain a mixture of inactivated polio virus of strains Mahoney, MEF1 and Saukett.
Although an attenuated Human enterovirus C has been produced and used as an attenuated oral polio vaccine, the attenuated Human enterovirus C may be dangerous because of the possible reversion of pathogenicity (paralysis-based neurovirulence) in persons administered to, or in contact with, whole viruses. Hence, there is a need for a safe and effective polio vaccine which is free of such pathogenicity.
Like all enteroviruses, four different Human enterovirus C coat/capsid polypeptides have been identified and are designated as VP1, VP2, VP3 and VP4, which associate to form an icosahedral virus capsid. Typically, vaccination with the individual polypeptides of Human enterovirus C has shown that the isolated polypeptides are not capable of raising neutralizing antibodies in humans and animals (Meloen, et al., J. Gen. Virol. 45:761-763, 1979).
U.S. Pat. No. 4,508,708 teaches that individual polypeptides of polio virus and hand, foot and mouth disease virus, VP1, VP2, VP3 and VP4, are not capable of raising neutralizing antibodies in humans and animals and that, among the individual polypeptides of the hand, foot and mouth disease virus, only VP1 possesses this capability. U.S. Pat. No. 4,508,708 demonstrates that, among the Human enterovirus C type 2 MEF1 virion VP1, VP2 and VP3 polypeptides, only the VP3 is capable of inducing neutralizing antibodies, although the antibody titer is low. It was found, however, that VP1, VP2 and VP3 are capable of inducing neutralizing antibodies only when the immunization is carried out with a preparation containing arildone, a broad spectrum antiviral agent that has been shown to selectively inhibit replication of picornaviruses (Langford, et al. Antimicrobial Agents and Chemotherapy 28:578-580, 1985).
Thus, the problem to be solved is the preparation of an effective vaccine which provides protective immunity against a human enterovirus infection, and without the use of antiviral compounds. The human enteroviruses for which protection is desired are, for example Human enterovirus A, including Coxsackievirus A16 and Human enterovirus 71; Human enterovirus B, including Coxsackievirus B serotypes, echoviruses and enterovirus serotypes; Human enterovirus C, including Human poliovirus 1, Human poliovirus 2 and Human poliovirus 3; as well as Human enterovirus D, including EV 68.
For the purposes of the instant invention, a vaccine is understood by those skilled in the art and may further be defined as a prophylactic or therapeutic material containing antigens derived from one more pathogenic organisms which, upon administration to a human subject or animal, will stimulate active immunity and protect against infection with these or related organisms (i.e., produce protective immunity).
Furthermore, protective immunity may be well understood by those skilled in the art. Nonetheless, protective immunity comprises, at least, the induction, or elicitation of neutralizing antibodies and/or T-cell immune response which will neutralize the virus.
It is well recognized in the vaccine art, that it is unclear whether an antigen derived from a pathogen will elicit protective immunity. Ellis (Chapter 29 of Vaccines, Plotkin, et al. (eds) WB Saunders, Philadelphia, at page 571,1998) exemplifies this problem in the recitation that “the key to the problem (of vaccine development) is the identification of that protein component of a virus or microbial pathogen that itself can elicit the production of protective antibodies . . . , and thus protect the host against attack by the pathogen.”
An approach to making improved vaccines against picornaviruses would be to mimic the virus capsid structure or its components which may elicit protective antibodies such as are produced with a killed whole virus vaccine. This kind of approach is safer than a killed, inactivated or attenuated vaccine approach because there is no opportunity for reversion.
All picornaviruses share the same genomic structure, including 4 structural genes within the P1 gene: VP1, VP2, VP3, and VP4, the VP4 and VP2 being expressed together as VP0, and viral proteases within the 3C and 3D genes. The viral protease will cleave the P1 gene, thereby allowing the virus to assemble into virus like particles (VLPs), virus capsomers, complexes and/or antigens of enteroviruses.
Vaccines have been proposed with indifferent success. It has been proposed to use subunit vaccines comprising the major capsid protein, VP1, of enteroviruses, as the basis of vaccines for the prevention and treatment of enterovirus infections, including HEV71 infections (Wu, et al., 2001).
With regard to prophylaxis against enterovirus infection, it is possible to envisage a killed virus vaccine approach, which has been shown to elicit protective antibodies. The low virus titres achieved in general makes manufacturing of such enterovirus vaccines a challenge.
The present invention pertains to vaccines including antigenic coat/capsid proteins of viruses of the Picornaviridae family and which vaccines are devoid of virus RNA which may contribute to neurovirulence. The vaccines of the invention may comprise as antigens, the polypeptides P1 or VP0, or capsid proteins (VP's), designated as VP1, VP2, VP3 and/or VP4, or immunologically or biologically active fragments thereof which elicit neutralizing antibodies against enteroviruses.
The present invention relates to vaccines in which the picornavirus antigens are present in the form of one or more picornavirus polypeptides, especially human Enterovirus peptides VP2, VP3 or VP0, immunogenic fragments thereof, and/or antigenic determinants thereof. The picornavirus polypeptides may be obtained by chemical synthesis or by means of recombinant DNA techniques using known human Enterovirus amino acid or nucleic acid sequences.